Phase-Variable Glycosylation in Nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a leading cause of respiratory tract infections worldwide and continues to be a global health burden. Adhesion and colonization of host cells are crucial steps in bacterial pathogenesis, and in many strains of NTHi, the interaction with the host is mediated by the high molecular weight adhesins HMW1A and HMW2A. These adhesins are N -glycoproteins that are modified by cytoplasmic glycosyltransferases HMW1C and HMW2C. Phase variation in the number of short sequence repeats in the promoters of hmw 1 A and hmw 2 A directly affects their expression. Here, we report the presence of similar variable repeat elements in the promoters of hmw 1 C and hmw 2 C in diverse NTHi isolates. In an ex vivo assay, we systematically altered the substrate and glycosyltransferase expression and showed that both of these factors quantitatively affected the site-specific efficiency of glycosylation on HMW-A. This represents a novel mechanism through which phase variation can generate diversity in the quantitative extent of site-specific post-translational modifications on antigenic surface proteins. Glycosylation occupancy was incomplete at many sites, variable between sites, and generally lower close to the C-terminus of HMW-A. We investigated the causes of this variability. As HMW-C glycosylates HMW-A in the cytoplasm, we tested how secretion affected glycosylation on HMW-A and showed that retaining HMW-A in the cytoplasm indeed increased glycosylation occupancy across the full length of the protein. Site-directed mutagenesis showed that HMW-C had no inherent preference for glycosylating asparagines in NxS or NxT sequons. This work provides key insights into factors contributing to the heterogenous modifications of NTHi HMW-A adhesins, expands knowledge of NTHi population diversity and pathogenic capability, and is relevant to vaccine design for NTHi and related pathogens.